METHOD FOR DIAGNOSING CARCINOMAS USING IRGD AND MAGENTIC RESONANCE TOMOGRAPHY (MRT)

Abstract

The present invention relates to an imaging diagnostic method, comprising a contrast agent including a contrast enhancer in a magnetic resonance imaging (MRI) assisted diagnosis of carcinoma diseases or carcinomas, in particular of hepatocellular carcinoma (HCC). The contrast agent is preferably a gadolinium compound, preferably Gd-DTPA including iRGD as a contrast enhancer, for the improved imaging of carcinomas, in particular HCC, in MRI. The invention furthermore relates to a method for the risk stratification of patients and subjects using the aforementioned diagnosis.

Claims

1-15. (canceled)

16. An imaging method for the diagnosis and/or risk stratification of carcinoma diseases on a patient, characterized in that, by way of a) a contrast agent comprising a free lanthanide compound, and b) a contrast enhancer iRGD c) a magnetic resonance imaging process is carried out.

17. The imaging method for the diagnosis and/or risk stratification of carcinoma diseases on a patient according to claim 16, characterized in that, by way of a) a contrast agent comprising a free lanthanide compound, or a) a contrast enhancer iRGD, b) a first magnetic resonance imaging process is carried out and with delay, by way of c) a contrast agent comprising a free lanthanide compound and d) a contrast enhancer iRGD, e) a second magnetic resonance imaging process is carried out.

18. The imaging method for the diagnosis and/or risk stratification of carcinoma diseases on a patient according to claim 17, wherein the second magnetic resonance imaging process takes place within 10 minutes to 6 hours, in particular 1 to 24 hours, in particular within several days, in particular within 4 to 24 hours after the first MRI.

19. The imaging method for the diagnosis and/or risk stratification of carcinoma diseases on a patient according to claim 16, characterized in that the carcinoma is selected from the group consisting of solid malignant neoplasms, breast cancer, colon cancer, pancreatic cancer, stomach cancer, ovarian cancer, biliary duct cancer, prostate cancer, cervical cancer, glioblastoma, bronchial cancer, pancreatic cancer, prostate cancer, renal cell cancer, bladder cancer, sarcomas, hepatocellular carcinoma (HCC), brain tumors, and tumor metastases of these cancers.

20. The imaging method for the diagnosis and/or risk stratification of carcinoma diseases on a patient according to claim 16, characterized in that the iRGD is selected from the group consisting of the sequences of CRGDKGPDC (SEQ II) NO: 1), CRGDRGPDC (SEQ ID NO: 2), CRGDKGPEC (SEQ ID NO: 3), and CRDGRGPEC (SEQ ID NO: 4).

21. The imaging method for the diagnosis and/or risk stratification of carcinoma diseases on a patient according to claim 16, characterized in that the free lanthanide compound is a gadolinium compound, in particular a complex made up of gadolinium and a chelator, in particular wherein the chelator is selected from the group consisting of diethylenetriaminepentaacetic acid (DTPA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 2-[4-(2-hydroxypropyl)-7,10-bis(2-oxido-2-oxoethyl)-1,4,7,10-tetrazacyclododec-1-yl]acetate, or 2,2,2-(10-((2R,3S)-1,3,4-trihydroxibutane-2-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate, optionally comprising auxiliary agents, such as salt-forming agents, and in particular meglumine.

22. The method for the diagnosis and/or risk stratification according to claim 16, for the identification of patients who are at an increased risk of and/or have an unfavorable prognosis for carcinoma diseases.

23. The method for the diagnosis and/or risk stratification according to claim 16, wherein the patient is a symptomatic and/or asymptomatic patient, in particular an emergency patient.

24. The method for the diagnosis and/or risk stratification according to claim 16, for the therapy control of carcinoma diseases of a patient, in particular in intensive medicine or emergency medicine.

25. The method for the diagnosis and/or risk stratification of carcinoma diseases on a patient according to claim 16, for carrying out clinical decisions, in particular advanced treatments and therapies using drugs, in particular in intensive medicine or emergency medicine, including the decision to hospitalize the patient.

26. The method for the diagnosis and/or risk stratification of carcinoma diseases on a patient according to claim 16, for the prognosis, for early detection and detection by differential diagnosis, for assessment of the severity, and for assessment of the course of the disease concomitant with the therapy.

27. A contrast agent comprising: a) a free lanthanide; and b) a contrast enhancer iRGD for use in an imaging method for the diagnosis and/or risk stratification of carcinoma diseases on a patient, wherein magnetic resonance imaging is carried out.

28. The contrast agent according to claim 27, wherein, by way of a) a contrast agent comprising a free lanthanide compound, or a) a contrast enhancer iRGD, b) a first magnetic resonance imaging process is carried out and with delay, by way of c) a contrast agent comprising a free lanthanide compound, and d) a contrast enhancer iRGD e) a second magnetic resonance imaging process is carried out.

29. The contrast agent according to claim 27, wherein the iRGD is intravenously applied.

30. A kit comprising one or more injection solutions for carrying out the method according to claim 16, comprising independently of one another, collectively or respectively a contrast agent, in particular Gd-DPTA, and/or a contrast enhancer iRGD at a dose of 0.1 to 12 mol/kg, 2 to 10 mol/kg, in particular 4 mol/kg body weight.

Description

EXAMPLES AND FIGURES

1. Peptides

[0059] Synthetic peptides iRGD (CRGDKGPDC) and the RGD control peptide (CRGDDGPKC), which have in a circular shape over a cysteine-cysteine disulfide bond between AS 1 and 9 (Sugahara et al., 2010), were acquired from GenScript USA Inc. with a purity of more than 98%.

2. Creation of TGF/c-Myc Transgenic Mice and Visualization of the HCCs

[0060] Male TGF/c-myc bitransgenic mice were created by crossing homozygous metallothionein/TGF and albumin/c-myc single-transgenic mice in a CD13B6CBA background as described (Murakami et al., 1993; Haupenthal et al., 2012). After weaning, the animals were given ZnCl.sub.2 by way of the drinking water to induce tumors via the expression of TGF. Starting at an age of 20 weeks, the TGF/c-myc animals underwent a Primovist-enhanced MRI scan in a 3T MRI Scanner (Siemens 113 Magnetom Trio, Siemens Medical Solutions) as described (Haupenthal et al., 2012; Watcharin et al., 2015; Korkusuz et al., 2013).

[0061] Mice with HCC according to the Primovist-enhanced MRI underwent the following experiments.

3. Creation of the HCC Nude Mice

[0062] HepG2- and Huh7 cells (ATCC and RIKEN BioResource Center) were cultivated in DMEM with 10% FBS and penicillin/streptomycin (Life Technologies). 5 million cells in 100 L PBS were injected into the sides of NMRI Foxn1 nude mice (Harlan Laboratories B.V.). Four weeks later the mice were assigned to the test groups.

4. 4T1 Breast Cancer Mouse Model

[0063] 4T1 cells (ATCC) were cultivated in RPMI 1640 medium with 10% FBS and penicillin/streptomycin (Life Technologies). BALB/c mice were injected with 2.5104 4T1 cells into mammary gland no. 4 of the mouse, and the tumors were allowed to grow for two weeks.

5. Gd-DTPA-Enhanced MRI with and without iRGD

[0064] So as to determine the effect of iRGD on the Gd-DTPA-enhanced MRI in the HCC, TGF/c mice with HCC according to a prior Gd-EOB-DTPA (Primovist)-enhanced MRI one week earlier, or nude mice with HepG2 or Huh7 tumors, were included in the experiments. The mice were anesthetized by way of intraperitoneal injection of ketamine (70 mg/kg body weight) and xylazine (10 mg/kg body weight), followed by a basal T1-weighted MRI. Directly thereafter, a Gd-DTPA-enhanced MRI was conducted (Haupenthal et al., 2012). Twelve to 24 hours later, either iRGD or the RGD control peptide was injected (100 mL each via the tail vein), followed by a basal and a Gd-DTPA-enhanced MRI (Watcharin et al., 2015). For the quantitative analysis of the MRI data, the signal intensities in user defined regions of interest (ROI) were used (Korkusuz et al., 2013). ROIs were placed in the liver and in the tumor tissue. The changes in the signal intensities were ascertained by subtraction of the pre-contrast value from those after the administration of Gd-DTPA. The changes in the signal intensities of the tumors and the livers due to iRGD or the RGD control peptide were indicated as multiples of the values of the Gd-DTPA MRI with prior injection of PBS.

Description of the Results:

[0065] Co-administration of iRGD, however not of a peptide containing an RGD motif, not however a CendR motif, selectively increases the penetration of the Evans blue dye and doxorubicin in carcinomas, in particular HCC.

[0066] So as to examine whether intravenously applied iRGD increases the permeability of the HCC, the effect of intravenously applied iRGD on the levels of co-injected Evans blue, an albumin-binding dye, in TGF/c-myc mice with endogeneous HCCs according to a Gd-EOB-DTPA-enhanced MRI were analyzed. For this purpose, the mice with HCC were intravenously injected with iRGD, an RGD control peptide without a CendR motif, or PBS, followed by the injection of Evans blue 15 minutes later. Another 30 minutes later, the animals were terminally perfused. The photometric quantification of the dye showed a three-fold increase in the Evans blue quantity in the tumors of iRGD-injected animals compared to HCCs of mice that had been injected with PBS (p=0.012) or the control peptide (p=0.012), while the RGD control peptide or PBS had no effect (FIG. 1A). iRGD or the RGD control peptide had no impact on the concentration of Evans blue in normal tissue (liver, kidney, spleen and lung), which supports the fact that iRGD specifically increases the permeability of the malignant tumor tissue of the HCCs in TGF/c-myc mice for co-applied substances.

[0067] So as to examine whether the tumor-permeabilizing effect of iRGD also occurs in HCC nude mice tumor models, the effect of intravenously applied iRGD on the level of co-applied Evans blue in nude mice which had HepG2 or Huh7 xenotransplants was analyzed. iRGD effectuated an increase in the concentrations of co-applied Evans blue in HepG2-xenotransplanted and Huh-7-xenotransplanted tumors by a factor of 3.4 (p0.002) (in HepG2 tumors) and 2.6-fold in Huh7 tumors (p<0.001) compared to the corresponding tumors of nude mice injected with PBS and the control peptide (FIGS. 1B and C).

[0068] Furthermore, the effect of iRGD on the tissue concentrations of intravenously applied doxorubicin, a tumor therapeutic agent that can be detected due to the inherent fluorescence in tissue sections and tissue extracts, in the TGF/c-myc and in the HepG2 xenotransplant HCC mouse model was analyzed. As is shown in FIG. 2, iRGD caused an increase in the doxorubicin level in the HCCs in both tumor models (p0.0011). iRGD had no effects on the levels of doxorubicin in the organs (FIG. 2A). The control peptide had no effects on the doxorubicin level in any of the tissues.

[0069] It was examined whether the tumor-permeabilizing effect of iRGD can already be detected by a Gd-DTPA-enhancing MRI, a widely used clinical procedure, in TGF/c-myc mice with endogenous HCC. For this purpose, TGF/c-myc mice with HCCs discovered one week prior according to Gd-EOB-DTPA-enhanced MRI were used. These animals received a Gd-DTPA-enhanced MRI, which showed highly negative contrasts of HCC in the liver (FIG. 3B, left). 12 to 24 hours later, the mice were administered iRGD or the RGD control peptide, followed by another Gd-DTPA-enhanced MRI. As is shown in FIG. 3A (left), the tumors of mice that had been injected PBS or the RGD control peptide were negatively contrasted. The injection of iRGD prior to the Gd-DTPA-enhanced MRI caused a substantial increase in the signal intensities of the tumors, which were now contrasting positively in the liver (FIG. 3A, right). iRGD and the RGD control peptide had no effects on the signal intensities in normal organs. The quantitative densitometric analysis of the signal intensities showed a 2-fold increase in the tumors following the injection of iRGD compared to animals who had been administered the RGD control peptide (p=0.004) or PBS (p<0.001; FIG. 3B).

[0070] Next, it was examined whether iRGD also influences the MRI signal in HCCs in HCC xenotransplant nude mice models. As is shown in FIG. 3C, iRGD caused a rise in the signal intensity of the tumors in HepG2 tumors (p=0.0311). Similar circumstances applied to Huh7 xenotransplants (p=0.0081), but the rise was less pronounced (1.5-fold; FIG. 3D) compared to the other HCC mouse models (2-fold). This data shows that the iRGD-induced rise in tumor permeability was able to be observed in all three different HCC mouse models by way of this non-invasive method (GD-DTPA-enhanced MRI without and with iRGD).

[0071] Furthermore, it was examined to what extent iRGD also influences the MRI signal of the tumor in the Gd-DTPA-enhanced MRI in the syngeneic 4T1 breast cancer mouse model. It was shown that iRGD resulted in a significant increase in the MRI signal in the Gd-DTPA-enhanced MRI in the 4T1 tumor (p<0.05).

[0072] The contrast enhancer iRGD thus allows a differential diagnosis so as to distinguish a benign change from a cirrhotically changed liver.

[0073] At present, the different blood supply of the liver and the tumor tissue primarily via the portal vein (liver) or the hepatic artery (HCC) for the radiological diagnostics of the HCC, which results in a delayed inflow of intravenously applied contrast agent in the liver compared to the HCC tissue, and the different distribution of anion transporters between the HCC and normal liver tissue. The latter is achieved by the distribution of the liver-specific contrast agent Primovist in the MRI.

[0074] FIG. 1:

[0075] iRGD, but not with the control peptide without a CendR motif, increased the concentration of systemically co-applied Evans blue (EB) in the HCCs in TGF/c-myc mice and in 2 HCC transplant nude mice models.

[0076] TGF/c-myc mice with MRI-verified HCCs (A) or mice with subcutaneous HepG2 (B) or Huh7 xenotransplants (C) were intravenously injected with 4 mmol/kg iRGD or control peptide (each in PBS), or PBS was injected alone, followed by an injection of EB 5 minutes later. The tissues were harvested after another 30 minutes. The EB accumulation in the tissues was determined by way of photometry. The data involves mean valuesSD; n=4 to 5. Asterisks indicate a significant difference (* P<0.05; * P<0.01; *** P<0.001). n.s., not significant.

[0077] FIG. 2:

[0078] The co-treatment with iRGD selectively increased the concentrations of doxorubicin in HCCs of TGF/c-myc mice and mice with HepG2 xenotransplants.

[0079] TGF/c-myc mice with radiologically verified HCCs (A) or nude mice with subcutaneous HepG2 xenotransplants (B) were intravenously injected with 4 mmol/kg iRGD or control peptide (each in PBS), or treated with PBS alone, followed by a doxorubicin (20 mg/kg iv) injection 10 minutes later. The tissues were collected another 30 minutes later and stored, and the doxorubicin concentration was determined. The values are mean valuesSD; n=5 to 6. Asterisks indicate a significant difference (**P<0.01; ***P<0.001).

[0080] FIG. 3:

[0081] iRGD resulted in a tumor-specific increase in the signal intensity in the Gd-DTPA-enhanced MRI. TGF/c-myc mice in which HCCs had been detected one week prior by way of Gd-EOB-DTPA-enhanced MRI were anesthetized, followed by a basal T1-weighted MRI, and a Gd-DTPA-enhanced MRI directly thereafter. 12 to 24 hours later, either iRGD or RGD control peptide (con. peptide) was injected (100 L each via the tail vein, 4 mmol/kg) into the same animals, followed by a basal MRI and a Gd-DTPA-enhanced MRI. A. Gd-DTPA-enhanced MRI of TGF/c-myc mice with HCC (left), followed by an injection of iRGD and another Gd-DTPA-enhanced MRI 12 hours thereafter. Arrows mark the tumor.

[0082] B. quantitative analysis of the MRI data. Values are mean valuesSD; n=12.

[0083] C. 12 nude mice with HepG2 xenotransplants were injected on day 1 with the control peptide, followed by a Gd-DTPA-enhanced MRI. The next day, the animals were treated with iRGD, followed by a Gd-DTPA-enhanced MRI. The values are mean valuesSEM. D. 13 nude mice with Huh7 xenotransplants were subjected to the same procedure as in (C). The values are mean valuesSEM. Asterisks indicate a significant difference and (* P<0.05; **P<0.01; ***P<0.001).

[0084] FIG. 4:

[0085] Effect of iRGD and the RGD control peptide on the increase in the signal intensity in the Gd-DTPA-enhanced MRI in the liver and in the 4T1 tumors in BALB/c mice. BALB/c mice were injected with 2.510.sup.4 4T1 cells into mammary gland no. 4 of the mouse. Two weeks later, the mice were anesthetized, followed by a basal T1-weighted MRI, and a Gd-DTPA-enhanced MRI directly thereafter. 12 to 24 hours later, either iRGD or RGD control peptide (con. peptide) was injected (100 L each via the tail vein, 4 mmol/kg) into the same animals, followed by a basal MRI and a Gd-DTPA-enhanced MRI. The data was quantitatively analyzed. Values are mean valuesSD; n=6.

LITERATURE

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